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Vanilla Rhizobacteria as Antagonists against Fusarium oxysporum f. sp. vanillae.

Byline: Jacel Adame-Garcia, Mauricio Luna-Rodriguez and Lourdes Georgina Iglesias-Andreu

Abstract

Pathogens such as Fusarium oxysporum strongly affect the health of various agricultural crops like vanilla. However, despite significant economic losses caused by this pathogen there is no efficient method for its control. Therefore, we propose using rhizobacteria obtained from vanilla roots against F. oxysporum f. sp. vanillae. The results showed that there was no positive correlation between the antagonism expressed under in vitro conditions and those expressed under greenhouse conditions. The 16S rDNA gene analysis indicated that the bacterial genera tested corresponded to Sphingobacterium, Staphylococcus, Serratia, Psychrobacter, Pseudomonas and Stenotrophomonas.

The in vitro antifungal activity was evaluated using three culture media (Potato Dextrose Agar, Nutrient Agar and Czapek) using the empty box technique (antagonism). Isolates of Staphylococcus xylosus BAC-JAG15, Serratia sp. BAC-JAG4 and Stenotrophomonas sp. BAC-JAG1 showed 90% in vitro antagonism against F. oxysporum in the three media tested. In the greenhouse evaluation, plants treated with these isolates initially showed symptoms of chlorosis without developing characteristic symptoms of disease produced by F. oxysporum f. sp. vanillae. This demonstrates protection provided by rhizobacteria against infection from F. oxysporum f. sp. vanillae in vanilla plants.

Keywords: Biological control; Soil borne fungal pathogens; Antifungal activity; Vanilla

Introduction

Vanilla (Vanilla planifolia Jacks. ex. Andrew) has low genetic variability (Minoo et al., 2008) due to the clonal origin of crops and vegetative propagation, making it susceptible to a large number of diseases caused by Phytophthora sp., Calospora vanillae Massee, Sclerotium spp., Colletotrichum gloeosporioides (Penz.) Sacc. and Fusarium spp. (Bhai and Thomas, 2000; Summerell et al., 2003; Guzman, 2004; Talubnak and Soytong, 2010).

Fusarium oxysporum is the most important pathogen responsible for severe damage to the cultivation of vanilla, and has thus been the most studied genus. However, despite significant economic losses caused by the disease in vanilla, there has not, to date, been an efficient method for controlling this disease. Therefore, it is imperative to investigate the association of rhizosphere manipulation, which contains beneficial microorganisms, with its antagonistic properties that protect roots from harmful effects caused by soil pathogens (Weller, 1988).

The potential uses of plant-associated bacteria as biocontrol agents of plant diseases have been described (Rovira, 1965; Hallmann et al., 1997; Sturz and Nowak, 2000; Welbaum et al., 2004), of which the growth promoting rhizobacteria or PGPR are the most studied (Kloepper and Schroth, 1978). The PGPR colonize root surfaces and/or are closely adhered to the interface between the soil and roots, the rhizosphere (Kloepper and Schroth, 1978; Kloepper et al., 1999).

Bacteria benefit the plant by directly interacting with beneficial microorganisms and the host plant, and through indirect mechanisms such as antifungal activity (Berg, 2009). They can reduce the incidence or severity of plant disease by acting as biological control agents or by exhibiting antagonistic activity towards a pathogen (antagonists) (Beattie, 2006).

Bacteria provide effective protection to plants by employing multiple modes of action such as production of antimicrobial compounds, production of enzymes that interfere with fungal pathogenesis, competition for resources, induction of plant resistance and interactions among members of the microbial community (Beattie, 2006).

Although numerous studies have been focused on using biocontrol against Fusarium spp. for various crops, there have been few studies to control this pathogen using rhizobacteria in vanilla. Among studies on the biological control of F. oxysporum f. sp. vanillae (Fov) with different strains of bacterial species are those by Tombe et al. (1997) who determined the effectiveness of Pseudomonas fluorescens as a Fov antagonist. The results obtained by Bhai and Kumar (2008) showed that P. fluorescens was a more effective antagonist to Phytophthora meadii, Fov and Colletrotrichum vanillae than other bacteria such as Enterobacter agglomerans, and Bacillus spp. However, these studies do not indicate whether the strains used corresponded to microorganisms isolated from root cultures of vanilla or were strains tested on other crops. As an alternative, the use of native species has not been reported for the control of phytopathogenic fungi.

Based on the above, the hypothesis of the present study is that microbial populations in the rhizosphere of vanilla contain bacteria with antagonistic activities that can be used as biocontrol agents against F. oxysporum f. sp. vanillae. The main objective was to evaluate the antagonistic capacity of rhizobacteria associated with Vanilla planifolia Jacks. ex Andrew against F. oxysporum f. sp. vanillae under in vitro conditions and in soil inoculated with the pathogen.

Materials and Methods

Experimental Details and Treatments

Experimental material: The F. oxysporum strain used in this work corresponds to F. oxysporum f. sp. vanillae, which was isolated and reported to be highly pathogenic to vanilla (Adame-Garcia et al., 2011).

Collection and Isolation of Rhizobacteria

Bacterial isolates were obtained from vanilla plant roots from Papantla, Veracruz (20 21' 47.57" N, 97 30' 39.06" W), Mexico, a region having high production of this crop. Samples were placed in plastic bags, labeled and transported to the laboratory. Roots were cut into 5 mm fragments and 10 g was weighed out from each root. Each sample was placed into a sterile 250 mL Erlenmeyer flask to which was added 90 mL sterile phosphate solution (0.25 M KH2PO4). The solution was stirred at 120 rpm for 10 min in an orbital shaker (Lab Line). Then five dilutions were prepared (1:9), and 0.1 mL of the 10-5 and 10-6 dilutions were spread onto agar crab shell medium (3, 6 and 9%), which were incubated at 26 1C for 72 h.

Evaluation of Antifungal Properties

In vitro evaluation: For this evaluation the fungal vs. bacterial antagonism produced when grown in culture medium was used.

Antagonism through Competition, Fungi vs. Bacteria in Culture Medium

For this determination, the empty box method was used with three media: nutrient agar (NA), potato dextrose agar (PDA) and Czapek Dox. A completely randomized design with five replicates was used for which 100 uL of bacterial solution (1 x 108 UFC mL-1) was used and distributed uniformly in 20 mL for each of the three culture media studied. Once the agar solidified, a sample of mycelium was applied with a dissecting needle. All boxes (treatments and control with no bacteria) were incubated in an incubator (Felisa) at 251C. The radial mycelial growth was measured at 7 days post-inoculation. Data from in vitro tests were analyzed with StatView 5.0 using ANOVA and Tukey tests to determine differences among treatments.

Greenhouse Evaluations

Greenhouse evaluations were conducted in ASrsulo Galvan, Veracruz, Mexico, located 8 masl at 19 24' N and 96 18' W. The climate in this region is hot sub-humid, with an average annual temperature of 25.8C and an average annual rainfall of 1,017.7 mm.

A randomized block design was used with 5 replicates (cuttings) and 50 treatments (Fig. 1). All treatments were conducted in black plastic bags (10 x 15 cm) containing 500 g of soil and infected with 10 mL of an Fov spore suspension (106 spores mL-1) from cultures 12 days old cultured on PDA medium and incubated at 261C. To obtain the spore suspension, 2 mL of Tween 20 solution (20%) was added to each petri dish containing the mycelia. This solution was then decanted into a test tube containing 8 mL of sterile distilled water and then mixed with a vortex (MAXI-MIX II). From this solution, aqueous suspensions containing 106 spores mL-1 were prepared. A sterile solution of Tween 20 (20%) without fungal inoculum was used as a control.

Three types of bacterial inoculation were used: (1) cuttings immersed in bacterial suspension for 10 min, (2) cuttings immersed in bacterial suspension for 10 min and re-inoculated three days after planting, and (3) inoculation at the three days after planting. As controls, cuttings inoculated with bacterial isolates from soil not inoculated with Fov, and cuttings without bacterial or fungal inoculum were used. Bacterial solutions of 1 x 108 UFC mL-1 were used from cultures maintained for 24 h in NA medium.

The test was conducted two days after inoculation of the mycelia in the soil and at different times before planting. With a sterile razor, an incision of 1 cm in length was made in the roots and this portion of the cuttings was placed in contact with the soil. The cuttings were watered three times a week. Temperatures ranged from 26-28C during the day and 22-24C at night.

The damage level of the cuttings was evaluated at 60 days after planting, and four levels of damage were identified: (1) cuttings without symptoms, (2) cuttings with symptoms of chlorosis, (3) cuttings with rot, and (4) necrotic or dead cuttings. After the evaluation, Koch's postulates were applied to confirm the presence of the inoculated fungus.

The data were analyzed using Statistica 7.0. The nonparametric Friedman ANOVA and Kendall's Coefficient of Concordance were applied. For nonparametric sample contrasts, the Wilcoxon test was used.

Molecular Identification of Bacterial Isolates

For the molecular identification of isolates, DNA extraction was performed according to Cheng and Jiang (2006). DNA integrity was assessed using 0.8% agarose gel electrophoresis (TBE 0.5 X) in a horizontal chamber (CONSORT) at 100 V. The gels were stained in 100 mL of 1X TBE solution with 2 uL of ethidium bromide (10 mg mL-1) for 20 min, and then observed with a MicroBis photodocumentation system.

Bacterial isolates were selected for their antifungal properties and were applied by Luria plate streaking on Luria Bertani medium (LB). The plates were incubated at 271C in a bacteriological incubator (Felisa).

The amplification of the 16S rDNA gene was performed following Luna et al. (2013) and using 0.20 mM of dNTPs and similar thermal cycling conditions. The PCR products were purified using a ChargeSwitch(r) - Pro PCR Clean-up Kit (Invitrogen) following the manufacturer's instructions and then sequenced at the Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico (UNAM) using an Applied Biosystems Sequencer (model 391) and employing the BMB-CR oligonucleotide.

The sequence data were edited using BioEdit version 7.0.5.3 (Hall, 1999) and analyzed via the Basic Local Alignment Search Tool (BLAST) system (GenBank, National Center for Biotechnology Information (NCBI).

Phylogenetic and molecular analyses were conducted using MEGA version 5 (Tamura et al., 2011) with the Maximum Parsimony (MP) method. The MP tree was obtained using the Maximum Likelihood method was based on the Tamura-Nei model (Tamura and Nei, 1993). The bootstrap consensus tree was inferred from 1,000 replicates (Felsenstein, 1985).

Results

Isolation of Rhizobacterial Strains

Bacterial colonies were visible at 4 days after planting. A total of 116 bacterial isolates were obtained for evaluation of antifungal activity against Fov.

In Vitro Evaluation, Fungal Antagonism vs. Bacterial Culture in Medium

Only seven of the 116 bacterial isolates tested exhibited inhibition relative to the controls.

Analysis of the 16S rDNA gene identified these isolates as Sphingobacterium sp. BAC-JAG26 and BAC- JAG89, Staphylococcus xylosus BAC-JAG15, Serratia sp. BAC-JAG4, Psychrobacter sp. BAC-JAG39, Pseudomonas sp. BAC-JAG101, and Stenotrophomonas sp. BAC-JAG1.

Evaluation of the antifungal capacity of these strains in the three cultivation media (Fig. 2) revealed significant differences among the isolates (df=7, Pless than 0.0001). In PDA medium, strains of Serratia sp. BAC-JAG4 and Sphingobacterium sp. BAC-JAG26 reduced growth of Fov by 69% and 52%, respectively (Fig. 3a). However, in NA medium, antagonistic action increased slightly for Serratia sp. BAC-JAG4 to 75% and markedly for Sphingobacterium sp. BAC-JAG26 to 80% (Fig. 3b). Particularly interesting was S. xylosus BAC-JAG15 on PDA medium which induced an inhibition of only 32%, contrasting with 94% and 81% in NA and Czapek media, respectively (Figs. 3b, c).

In Czapek medium, some bacterial isolates showed a different trend compared to PDA and NA media. Particularly, Pseudomonas sp. BAC-JAG101, Sphingobacterium spp. BAC-BAC-JAG26 and BAC- JAG89, which inhibited Fov growth, showed no inhibition in Czapek medium (Fig. 3c). Psychrobacter sp. BAC-JAG39 had the lowest inhibition percentage (Fig. 3b) on PDA and Czapek media and had a slight stimulatory effect (3%) on fungal growth in NA medium.

Isolates S. xylosus BAC-JAG15, Serratia sp. BAC-JAG4 and Stenotrophomonas sp. BAC-JAG1 reduced the growth of the fungus on PDA and NA media by 94%, and also exerted an inhibitory effect in Czapek medium, by 81%, 77% and 79%, respectively (Fig. 3c).

Greenhouse Evaluation

The results of greenhouse evaluation (Fig. 4) revealed significant differences (df=9, p=0.00821) in the inhibition of Fov activity among bacterial isolates, when vanilla cuttings were immersed for 10 min in a bacterial suspension and then planted in soil inoculated with the fungus (Fig. 5a).

Psychrobacter sp. BAC-JAG39 expressed the greatest antifungal properties as there were no significant differences between it and the negative control treatment (Fig. 5a). However, when using other methods of inoculation (three days after planting), the treated cuttings showed signs of chlorosis. This result was also observed in treatments inoculated with Serratia sp. BAC-JAG4 (Fig. 5c). Treatments with no favorable results were those where the cuttings were inoculated with Pseudomonas sp. using any of the three methods of inoculation, with the cuttings presenting symptoms of chlorosis, including tissue rotting and death (Fig. 5a, b and c).

In treatments where the cuttings were inoculated with the different bacterial suspensions and planted in soil without Fov inoculation, significant differences were detected among them (df=9, p=0.0009) that were attributable to a pathogenic from bacterial strain. Such is the case when isolating Pseudomonas sp. where the cuttings, without the presence of Fov, developed chlorosis (Fig. 6a) and then symptoms of tissue rot (Fig. 6b).

In treatments where the cuttings were immersed in bacterial suspensions of Sphingobacterium spp., S. xylosus BAC-JAG15 and Serratia sp. BAC-JAG4, there were slight symptoms of chlorosis, but these treatments were not significantly different compared to the negative control (Fig. 6a and b). Furthermore, in treatments where the cuttings were inoculated three days after planting, these bacteria did not produce any damage to vanilla stems (Fig. 6c).

Discussion

In the present study, we used culture media with added crab shell as an alternative to selecting bacterial strains capable of producing extracellular enzymes to hydrolyze the chitin present in the cell walls of fungi (Chang et al., 2003; Wang et al., 2006; Chang et al., 2009; Wang et al., 2009; Wang et al., 2010) where certain strains of Bacillus cereus and B. subtilis, when grown in media with crab and/or shrimp shells, produce chitinolytic enzymes efficient for the in vitro inhibition of F. oxysporum, F. solani and Pythium ultimum.

Obtaining strains of Sphingobacterium, Psychrobacter, Pseudomonas, Serratia, Staphylococcus and Stenotrophomonas from other plant species using culture media with crab and/or shrimp shells as the only carbon source has been reported (Wolf, 2002; Chang et al., 2003; Ribbeck-Busch et al., 2005; Wang et al., 2006; Chang et al., 2009; Wang et al., 2009; Zachow et al., 2009). Likewise, strains of these genera have been reported as rhizobacteria with in vitro antagonistic activity against phytopathogenic fungi, including F. oxysporum, but not for the strain vanillae. To date, only the strains of Pseudomonas sp. have been used as antagonists of Fov (Tombe et al., 1997; Bhai and Kumar, 2008).

Here, the results from Serratia sp. BAC-JAG4, which reduced fungal growth in the three culture media tested and did not yield symptoms of wilt or rot, are consistent with previous studies reporting that S. liquefaciens, S. plymuthica and S. rubidaea showed in vitro antifungal activity against different fungal pathogens (Kalbe et al., 1996). In particular, isolates of S. plymuthica have been used as biocontrol agents against Verticillium dahliae, Rhizoctonia solani and F. oxysporum in wheat, oats, cucumber, corn, canola and potato (Astrom and Gerhardson, 1988; Kloepper et al., 1992; Kalbe et al., 1996; Berg, 2000; Frankowski et al., 2001; Ting et al., 2011).

The antifungal mode of action for Serratia is based on antibiosis (prodigiosin and pyrrolnitrin production) and production of degradative enzymes of fungal cell walls (chitinases and b-1, 3 glucanase). As well, they produce potent siderophores to improve iron availability. However, it has been shown that the mode of action is specific to each species of Serratia (Kalbe et al., 1996; Guevara-Avendano et al., 2014). For example, S. liquefaciens and S. marcescens produce chitinases similar to those of S. plymuthica which has been used to inhibit the germination of spores of the phytopathogenic fungus Botrytis cinerea (Frankowski et al., 2001).

Another bacterial isolate that provided antagonism in the three media (PDA 32%, NA 94%, Czapek 81%) was S. xylosus BAC-JAG15. However, this species is commonly found in foods, as it is one of the main agents used for the fermentation of meat; it has not yet been reported as a biocontrol agent as only some strains may be potentially harmful and act as opportunists in animal infections (Schleifer and Kloos, 1975; Dordet-Frisoni et al., 2007). Some species of Staphylococcus possess antagonistic activities, such as S. epidermidis 2P3-18 isolated from the potato phyllosphere which presents some inhibition against V. dahliae and R. solani (Berg et al., 2005; Kai et al., 2007).

S. xylosus BAC-JAG15 requires more evaluation, since even some strains of this species, as well as those of Pseudomonas, Enterobacter, Azotobacter and Azospirillum produce growth regulators such as ethylene, auxins and cytokinins, which can promote plant growth and induce systemic resistance against pathogens (Arshad and Frankenberger, 1991; Leifert et al., 1994; Berg and Hallmann, 2006; Bhai and Kumar, 2008).

Another bacterial genus isolated from the roots of vanilla is Stenotrophomonas, its importance lies in its ecological functions within the cycles of elements in nature, its potential to promote plant growth and its applications in the biological control of plant fungal diseases (Ikemoto et al., 1980; Berg et al., 1994; Nakayama et al., 1999; Kobayashi et al., 2002). Stenotrophomonas sp. BAC-JAG1 showed antagonism against Fov in the three media tested (PDA 20%, NA 70% and Czapek 80%).

Similar results have been obtained in previous studies such as with Stenotrophomonas rhizophila and S. maltophila isolated from the rhizosphere of canola and potatoes showing antifungal properties, and the latter species showing high variability in its antifungal activity (Berg et al., 1999; Minkwitz and Berg, 2001; Wolf, 2002). It has been suggested that the mechanisms of inhibition of fungal isolates of the genus Stenotrophomonas are species dependent. These involve the production of secondary metabolites such as antibiotics and siderophores and enzymes to degrade the cell wall of fungi (proteases, glucanases and chitinases) (Minkwitz and Berg, 2001).

Apart from the genera described above, Sphingobacterium spp. BAC-JAG26 and BAC-JAG89 showed antifungal activity against Fov in inhibiting mycelial growth in two of the three media tested (PDA 52% and NA 80%). It is of interest to note that Sphingobacterium multivorum KST-009, from soil with added chitin, showed activity of a degrading enzyme for chitosan (chitosan SM1). Evaluation of germinated spores of F. oxysporum showed morphological changes such as protuberances in the hyphae which prevented cell differentiation at the ends of the hyphae and contributed to stopping or slowing mycelial elongation (Matsuda et al., 2001). These results agree with those obtained by Sturz et al. (1999) who reported on the antifungal ability of the bacterial species Sphingobacterium thalpophilum and Psychrobacter immobilis against Fusarium sambucinum, F. avenaceum and F. oxysporum to slightly inhibit in vitro growth.

Although Pseudomonas species have been widely studied for their antifungal capacity, there are insufficient studies on this in vanilla. The work of Tombe et al. (1992) and Bhai and Kumar (2008) reported isolates of Pseudomonas fluorescens with in vitro activity against Fov. Tombe et al. (1997) included evaluations of greenhouse isolates of P. fluorescens which showed in vitro antifungal activity and concluded that these bacteria are effective at reducing the occurrence of rot when the stems or roots of vanilla were submerged in bacterial suspension before being transplanted into soil infested with Fov. Bhai and Kumar (2008) reported that Fov infection can be controlled by inoculating vanilla plants with isolates of P. fluorescens together with isolates of Bacillus sp. and B. polymyxa.

The results obtained in the present study clearly showed the differences in the quality and effectiveness of the bacterial isolates tested. The effect expressed by them in the greenhouse was different from that observed in vitro. These results agree with those reported by Knudsen et al. (1997) who noted that there is no correlation between the antagonism shown by bacterial isolates in vitro and those under greenhouse conditions. In the present work, most of the stems treated with bacterial inoculum initially developed symptoms of infection, but the disease did not advance, while in the control (cuttings without bacterial inoculum) infection spread from the root until the plant collapsed over a period of 30 days. These results demonstrate the protection offered by rhizobacteria at controlling Fov infection.

Cases such as Psychrobacter sp. isolate BAC-JAG39 had the lowest in vitro inhibition, had greenhouse antifungal properties in the three types of tested inoculations. The observed failure in controlling the antifungal activity observed in the greenhouse or the presence of chlorosis in vanilla stems may be due to problems with the procedure for proper establishment of bacterial isolates. It is known that environmental factors can result in poor distribution, insufficient development of strains in roots or poor expression of antagonistic activity by bacterial isolates (Duffy and Weller, 1995).

As well, some studies (Radjacommare et al., 2010) have shown that care must be taken to generalize conclusions in such studies, since it is not known to what extent biocontrol activity will be affected by biotic factors (species and variety of plant, soil microbial activity), abiotic factors (soil type, water potential, soil temperature) and other factors (method and frequency of activities). Bhai and Kumar (2008) have emphasized that the degree of in vitro inhibition and the potential of rhizobacteria for suppression of Fov infection varies according to the type of insolate used.

The bacterial isolates used in this study were selected from culture media supplemented with chitin (crab shells). Future studies are needed to evaluate the antifungal activity of these isolates by adding the same ground chitin wastes or other sources of chitin, and verifying the effectiveness of biocontrol. Previous studies have indicated that application of P. fluorescens formulations with added chitin can raise the efficiency of antagonism to increase the capacity to degrade chitin in pathogen hyphae cell walls (Radjacommare et al., 2010).

The greenhouse results show that all in vitro evaluations of biocontrol agents should be confirmed under field conditions, as there is no guarantee that candidates secreting toxins into the in vitro culture media and that cause pathogen inhibition will do so under field conditions. As the production of these secondary metabolites is known, results depend largely on the nutritional and specific environmental conditions (Kohl et al., 2011), results that have been demonstrated under the in vitro conditions of the present study, showing differences in antagonism depend on the culture medium used.

Acknowledgements

To the Consejo Nacional de Ciencia y Tecnologia (CONACyT) and Tecnologico Nacional de Mexico (TecNM), for providing a grant (27314) and license for a grant study assigned to the first author for her doctoral studies.

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Author:Adame-Garcia, Jacel; Luna-Rodriguez, Mauricio; Iglesias-Andreu, Lourdes Georgina
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:1MEX
Date:Feb 29, 2016
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